Every year, over 12,000 Americans succumb to metastatic renal cell carcinoma (MRCC) [1]. The prognosis of this disease is poor and the median survival time is only 1-2 years [2]. Chemotherapy is ineffective [3] with the notable exception of rare tumors with sarcomatoid features [4]. Until recently, cytokine-based immunotherapy was the only effective therapeutic approach, however, response rates were low and the treatment was accompanied by substantial side effects. Identification of reliable predictors of response and survival are necessary to choose patients who most likely benefit from these drugs. This question is now even more pertinent since the approval of sorafenib, sunitinib and temsirolimus has expanded the therapeutic options available to MRCC patients. Currently clinical and laboratory information may distinguish different groups [5], but molecular information such as protein [6, 7] or genetic data [8, 9] may further improve pre-therapeutic risk assessment.
A Single Nucleotide Polymorphism (SNP) is a variation in the DNA sequence, which occurs when a nucleotide (A, T, C or G) is changed in at least 1% of a certain population. When a SNP falls in a coding sequence, it may determine a change of an amino acid in the related protein sequence. Such a SNP is called non-synonymous. In accordance with the degeneracy rules of the genetic code, a SNP could also generate the same amino acid, which is than called a synonymous SNP. Of note, several studies have indicated that a SNP in a non-coding region of a gene may also impact biological processes.
SNPs in the human genome contribute to wide variations in how individuals respond to medications, either by changing the pharmacokinetics of drugs or by altering the cellular response to therapeutic agents [10]. Several studies have assessed the importance of these SNPs in predicting prognosis and response to therapies and drugs. For example, SNPs have been associated with prognosis of breast cancer, lymphoid neoplasms, and nasopharyngeal carcinoma [1]-17]. In RCC, Ito et al. [8] found that a SNP in the Signal Transducer and Activator 3 gene (STAT3) is associated with a greater likelihood of response to interferon-alpha.
The carbonic anhydrase 9 gene (CA9) is located on chromosome 9p12-13, which represents a chromosomal area linked to prognosis in RCC [18-20]. CA9 comprises 11 exons and encodes for the 459 amino acid protein CAIX. CAIX is a membrane associated protein and catalyzes the reversible reaction H2O+CO2H++HCO3−, which is crucial to a wide variety of processes including pH regulation. CAIX is not expressed in the majority of benign organs and tissues, but abundantly expressed as a direct consequence of hypoxia in numerous cancers [21]. Studies demonstrate that high CAIX expression in clear cell RCC is associated with better prognosis and a greater likelihood of response to IL-2 based immunotherapy [22, 23]. Taken together, CA9 is located in a prognostically relevant chromosomal area and is encoding for one of the most significant protein markers in metastatic RCC. In contrast to CAIX protein, however, no efforts have been made to date to study the CA9 gene in metastatic RCC. Here, we test the hypotheses that SNPs and mutations of the CA9 gene are associated with CAIX expression, response to immunotherapy and survival in metastatic rCC.
At present, there are several FDA-approved drugs available for the treatment of MRCC, namely IL-2, sunitinib, sorafenib, and temsirolimus [39-41]. Pre-therapeutic prognostic assessment is required to select patients most likely to benefit from certain agents. However, only a few reliable predictors of response and survival are currently available. Motzer et al. [5] utilized clinical (performance status, time from diagnosis to start of therapy) and laboratory data (lactate dehydrogenase, hemoglobin, and corrected calcium levels) to predict survival of patients treated with interferon-alpha. Zisman et al. [42] stratified patients with MRCC into three prognostic groups based on stage, performance status and Fuhrman grade. Protein expression in the tumor and genetic information may further assist in prognostic assessment. Kim et al. [6] assessed 8 molecular markers in MRCC and found that CAIX, PTEN, p53, and vimentin expression significantly enhanced the predictive accuracy of a clinical prognostic model. Ito et al. [8] analyzed a cohort of 75 Japanese MRCC patients treated with interferon-alpha. They found that rs4796743, which is located in the non-coding 5′-flanking region of STAT3 gene, is associated with a 2.7 fold greater likelihood of response to interferon-alpha.
A leading treatment for MRCC is immunotherapy with IL-2. This treatment is associated with severe toxicities. Accordingly, there is a need to identify patients for whom IL-2 would be of sufficient benefit to warrant the health risks. This invention provides for this need by providing a means for identifying such patients.